Insulin is a protein hormone consisting of an acid A-chain of 21 amino acid residues and a basic B-chain of 30 amino acids. A chain and B chain are bonded together by six cysteine residues forming three disulfide bond between following positions: A 6-A 11; A 7-B 7; A 20-B 19 (FIG. 1).
The three-disulfide bonds are important in maintaining the native conformation and biological activities of the insulin molecule. Insulin folds into a unique three-dimensional structure mainly composed of three α-helical segments (A2-A8, A13-A19, and B9-B19) stabilized by its three disulfides bonds.
Insulin analogues and derivatives differ from human insulin at one or more than one amino acid positions and/or amino acid chain length.
Insulin, Insulin analogues and derivatives are prepared using recombinant DNA technology in E. Coli or yeast. When E. Coli is used as host cell, insulin expressed will not be in native soluble and biologically active conformation. Instead of native protein, inactive inclusion bodies accumulated in host cell. These inclusion bodies contain recombinant protein in a highly enriched form with incorrect folding. As a consequence, the recombinant protein must be isolated, refolded under suitable conditions, and enzymatically converted to the biologically active insulin.
There are two important issues in recovering active proteins from inclusion bodies. These include:                (a) Solubilization of proinsulins, and        (b) Refolding of proinsulins.        
The chaotropic agents and detergents are commonly used as solubilizing agents. They act as protein denaturant. The chaotropic agents break hydrogen bridges in solution, thus disrupting the inter-molecular and intra-molecular interactions with partial or complete unfolding of the protein structure.
A key to the solubilization process is the addition of a reducing agent to maintain cysteine residues in the reduced state and thus prevent non-native intra- and inter-disulphide formation in highly concentrated protein solutions at alkaline pH.
Refolding is accomplished by removal of excessive denaturants by dilution, buffer exchange, diafiltrations, gel filtration chromatography or immobilization onto the solid support. Because of its simplicity, dilution is usually preferred for industrial scale refolding of proteins.
The concentration of protein present in a solubilizing mixture containing reducing agent and chaotropic auxiliary plays an important role in deciding the final yield of correctly folded proinsulins. As the protein concentration in solubilizing media containing both is cysteine and chaotropic auxiliary is increased, the probability of aggregation or precipitation of proteins increases due to increased interaction.
The other factor that results in aggregation of protein molecules is the sudden change in denaturant concentration, which forces protein molecules to collapse into compact structure resulting in precipitation or aggregation.
When denaturant is removed during refolding the hydrophobic effect drives the unfolded protein molecule to sequester their hydrophobic groups, leading to aggregation. For industrial application it is desirable to eliminate or minimize the formation of protein aggregates.
U.S. Pat. Nos. 5,663,291; 5,473,049; 5,986,048; 6,380,355 and U.S. Patent Application 20070106063 disclose processes for obtaining a precursor of insulin or an insulin derivatives thereof having correctly bonded.
Winter, J. et al. Renaturation of human proinsulin-a study on refolding and conversion to insulin. Analytical biochemistry (2002), 310 (2), 148-155 discloses refolding of human proinsulin under suitable redox conditions.